Firefly toxin lucibufagins evolved after the origin of bioluminescence

Abstract Fireflies were believed to originally evolve their novel bioluminescence as warning signals to advertise their toxicity to predators, which was later adopted in adult mating. Although the evolution of bioluminescence has been investigated extensively, the warning signal hypothesis of its origin has not been tested. In this study, we test this hypothesis by systematically determining the presence or absence of firefly toxin lucibufagins (LBGs) across firefly species and inferring the time of origin of LBGs. We confirm the presence of LBGs in the subfamily Lampyrinae, but more importantly, we reveal the absence of LBGs in other lineages, including the subfamilies of Luciolinae, Ototretinae, and Psilocladinae, two incertae sedis lineages, and the Rhagophthalmidae family. Ancestral state reconstructions for LBGs based on firefly phylogeny constructed using genomic data suggest that the presence of LBGs in the common ancestor of the Lampyrinae subfamily is highly supported but unsupported in more ancient nodes, including firefly common ancestors. Our results suggest that firefly LBGs probably evolved much later than the evolution of bioluminescence. We thus conclude that firefly bioluminescence did not originally evolve as direct warning signals for toxic LBGs and advise that future studies should focus on other hypotheses. Moreover, LBG toxins are known to directly target and inhibit the α subunit of Na+, K+-ATPase (ATPα). We further examine the effects of amino acid substitutions in firefly ATPα on its interactions with LBGs. We find that ATPα in LBG-containing fireflies is relatively insensitive to LBGs, which suggests that target-site insensitivity contributes to LBG-containing fireflies' ability to deal with their own toxins.


Supplemental Figures
. Docking structure of core lucibufagin and bufalin on pig ATP1A1.Chemical structures of (A) bufalin and (B) core lucibufagin.The best docking structure of core lucibufagin-ATP1A1 complex, defined as the structure from the top 10 highest affinity dockings that was closest to the co-crystal coordinates of bufalin, shown only with core lucibufagin (C), or with both core lucibufagin and bufalin (D).The βsurface of core lucibufagin interacts with residues E117, E327, N122 and T797.Bufalin interacts with residues D121, E327, V322 and T797.Red: core lucibufagin; green: bufalin; yellow dotted lines: hydrogen bounds.

Fig. S5.
Structures of core lucibufagin binding pocket and molecular docking simulations of pig ATP1A1 carrying single amino acid substitution.(WT) pig ATP1A1 bound to core lucibufagin (in red).The docking position was inferred by the bufalin and pig ATP1A1 co-crystal structure (PDB: 4RES).Yellow dotted lines show possible hydrogen bonds between ATP1A1 and core lucibufagin.All ligand-protein interactions formed on the β-surface of core lucibufagin and bufalin are listed in Table S9.*Common fragments number less than five suggests the corresponding peak with matching molecular weight is not LBG.Table S9.Hydrogen bonds formed between bufalin or core lucibufagin with the pig ATP1A1 (PDB 4RES).
Table S10.Docking simulations of core lucibufagin to native ATPα proteins from firefly species.

Fig. S1 .
Fig. S1.Phylogenetic trees of bioluminescent beetles.Maximum likelihood trees of the 41 species inferred from concatenated nucleotide sequences (A) or concatenated protein sequences (B) of 1,353 single copy orthologs.The nucleotide tree used GTRGAMMAI model and 1,000 bootstrap replications.The protein tree used PROTGAMMALGX model and 100 bootstrap replications.The trees were rooted with the non-bioluminescent outgroup Lycocerus sp. in the Cantharidae family.Colored squares denote the geographic distribution of each species.

Fig. S3 .
Fig. S3.Ancestral state reconstructions of the LBGs across the bioluminescent beetles under "ER" model (left) and "ARD" model (right) using phytools.Inferred ancestral states are shown by pie charts, and the red and white portions denote the posterior probabilities of LBGs presence and absence, respectively.

Fig. S4
Fig. S4.Docking structure of core lucibufagin and bufalin on pig ATP1A1.Chemical structures of (A) bufalin and (B) core lucibufagin.The best docking structure of core lucibufagin-ATP1A1 complex, defined as the structure from the top 10 highest affinity dockings that was closest to the co-crystal coordinates of bufalin, shown only with core lucibufagin (C), or with both core lucibufagin and bufalin (D).The βsurface of core lucibufagin interacts with residues E117, E327, N122 and T797.Bufalin interacts with residues D121, E327, V322 and T797.Red: core lucibufagin; green: bufalin; yellow dotted lines: hydrogen bounds.

Fig. S6 .
Fig. S6.Maximum likelihood tree of CYP303 from 41 beetles suggests gene family expansion in LBGs-containing species (highlighted in colors).The fruit fly D. melanogaster CYP303 was used as an outgroup.ML Bootstrap values greater than 0.7 from 1,000 replicates are shown.

Fig. S7 .
Fig. S7.GC-MS analysis of terpinolene in firefly species.The chromatograms from top to bottom represent authentic terpinolene standard, extracts from adult male of Aquatica leii, larva of A. leii, Sclerotia flavida, Pygoluciola qingyu and Pyrocoelia analis, respectively.Terpinolene were only detected in larva of A. leii.

Fig. S8 .
Fig. S8.Parent ion of lucibufagin C and its fragments in mass spectra from Diaphanes citrinus sample.A. low energy channel; B. high energy channel.MS peak at m/z=533.2385Da is attributed to protonated lucibufagin C ([C28H37O10] + ).Other marked MS peaks are fragments of [C28H37O10] + .

Fig. S9 .
Fig. S9.Fragment assignment of protonated lucibufagin C from Diaphanes citrinus sample in high energy mass spectrum using UNIFI.

Fig. S10 .
Fig. S10.Structural alignment of predicted structures of native ATPα proteins from firefly species using AlphaFold2 and Modeller9, measured as heavy-atom root mean square deviation (RMSD) in Ångstrom.

Supplemental TablesTable S1 .
Sampling information of the 21 species collected in our study.
1 three newly identified species 2 preserved museum specimens obtained from the museum of biology, Sun Yat-sen University 3 two species with publicly available genomic data.

Table S2 .
Summary of data from 41 taxa used in our study.

Table S3 .
Summary statistics of de novo assembled transcriptomes.A.Samples sequenced in this study.B.Samples with publicly available RNAseq data.A．

Table S4 .
Precursor ion and common fragment ions analysis based on eight known LBG skeleton structures.

Table S5 .
Screening MS E mass data of each species for peaks matching the five LBGs common fragments.

Table S6 .
Screening the MS E mass data of each species for peaks matching the molecular weights with previously reported 29 LBGs.

Table S7 .
List of species that have been previously examined for LBGs.

Table S8 .
Three fossil calibrations used (A) and divergence time estimates with comparison to previous studies (B).Posterior means and 95% intervals (in parentheses) are in Mya.

Table S11 .
Docking simulations of core lucibufagin onto pig ATP1A1 carrying specific amino acid substitution